BAF155 promotes cardiac hypertrophy and fibrosis through inhibition of WWP2-mediated PARP1 ubiquitination

co-transfection


Ethics approval
The study included five male patients and five age-and gender-matched controls. Failing heart samples were obtained from patients with end-stage heart failure (average ejection fraction was 20 ± 5%) at the time of cardiac transplantation (Northern Theater Command General Hospital). Nonfailing hearts were obtained from donors who had normal cardiac contractile function by echocardiography or anatomical analysis of remains and had died from

Immunohistochemistry for human hearts tissues from normal and heart failure patients
Heart tissues were fixed in neutral buffered formalin solution, embedded in paraffin, and then cut into 5-um serial sections. Sections were dewaxed in xylene, rehydrated in a series of descending percentages of ethanol and boiled in Tris-EDTA solution for antigen retrieval.
UltraSensitiveTM SP IHC Kit (MXB, China) was used for immunostaining. After blocking, the slides were incubated with primary antibody for BNP (Abcam, USA) and BAF155 at 37°C for 1 h. After washing, the sections were immersed with biotin-conjugated IgG secondary antibody in the IHC kit for 20 min, then with DAB (MXB, China) for 2 min, and then stained with hematoxylin for 2 min. Immunohistochemical staining was semiquantitated by determining the percentage of cardiomyocytes stained using 10% increments (0%, 10%, 20%, 30%, …, 100%) as in a previous study 1 . The mean values from two evaluators were used for statistical analysis.

Protein extraction
Specimens were pulverized in liquid nitrogen and placed in 5-mL tubes, Supplementaryed four volumes of lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail) and sonicated three times on ice with a Scientz ultrasonic homogenizer. This was followed by centrifugationclearing at 12,000 g and 4°C for 10 min. Protein concentration of the resulting supernatant was measured using BCA kit.

Trypsin digestion
Equal quantity of total protein in each sample was enzymatically digested, and the volume was adjusted to be consistent across the sample set. TCA was added dropwise to a final concentration of 20%, followed by vortex mixing and precipitation at 4℃ for 2h. Centrifugation was performed at 4500 g for 5 min, and the resulting pellet was washes with pre-cooled acetone twice. Followed by drying of pellet, pellet was resuspended by 200mM TEAB facilitated by sonication, trypsin was added to each sample at 1:50 (protease: protein, w/w) for overnight digestion. Dithiothreitol (DTT) was added at 5 mM followed by incubation at 56℃ for 30 min.
Then, iodoacetamide (IAA) at 11 mM was added followed by incubation for 15 min at room temperature at darkness.

LC-MS/MS analysis
Tryptic peptides were dissolved in liquid chromatographic mobile phase A and separated on a NanoElute ultra-high performance liquid system. Mobile phases A and B were 0.1% formic acid and 2% acetonitrile in water and 0.1% formic acid in acetonitrile, respectively. Peptides were eluted using the gradient with a constant flow rate at 450 nL/min. The elution gradient was set as: 0-72 min, 7%-24% B; 72-84 min, 24%-32% B; 84-87 min, 32%-80% B; 87-90 min, 80% B. Peptides were separated on a capillary column (inner ID, particle size) before injecting into a capillary ion source for ionization and TIMS-TOF Pro mass spectrometer (ion source voltage, 1.6 kV; scanning range, 100-1700 Da). The parallel accumulation serial fragmentation (PASEF) mode was enabled for data acquisition. Precursors with charge states 0 to 5 were selected for fragmentation, and 10 PASEF MS/MS scans were acquired per cycle. The dynamic exclusion time of MS/MS scanning was 30 seconds to prevent multiple scanning of the same parent ions.

Database search
Raw mass spectrometry data was searched against a Swissprot protein sequence database (Mus_musculus_10090_SP_20201214.fasta) by Maxquant (v1.6.15.0) with reverse decoy entries and common contamination proteins. A maximum of 2 missing cleavages was allowed for Trypsin/P digestion, and at least 7 amino acids were required for each peptide. Mass error tolerance for precursor ion was 10 ppm and product ion was 20 ppm, respectively. Cysteine alkylation [carbamidomethyl (C)] was considered a fixed modification. Variable modifications were methionine oxidation and n-terminal acetylation. Lysine ubiquitination and di-glycine on lysine were also set as variable modification for corresponding modification enrichment analysis. The FDRs for protein and PSM identifications were both 1%.

Proteomics quantification (relative value)
The raw LC-MS datasets were first searched against database and converted into matrices containing LFQ intensity (the raw intensity after correcting the sample/batch effect) of proteins.
The LFQ intensity (I) was transformed to the relative quantitative value (R) after centralization.
The formula is listed as follow where i represents sample and j represents protein:

Differential screening of proteomics (fold change)
Firstly, the samples to be compared were selected in pairwise groups, and the fold change (FC) was then calculated by the ratio of the mean intensity for each protein in two sample groups. For example, to calculate the fold change between sample A and sample B, the formula is shown as following: R denotes the relative quantitative value of the protein, i denotes the sample and k denotes the protein.
FCA/B,k = Mean(Rik, i∈A) / Mean(Rik, i∈B) To calculate the statistical significance of difference between groups, the student's T test was performed on the relative quantitative value of each protein from the two sample groups.
The corresponding P value was calculated as the significance index. The relative quantitative value of proteins was applied with log2 transformation typically. The formula is shown as following: Pik = T.test(Log2(Rik, i∈A), Log2(Rik, i∈B)) The protein with P value < 0.05, the fold change > 1.5 was regarded as significantly upregulated protein, while the protein with P value < 0.05, the fold change < 1/1.5 was regarded as significantly down-regulated protein. (P value < 0.05, the fold change > 1.5).

Gene Ontology (GO) analysis
UniProt-GOA database (www.http://www.ebi.ac.uk/GOA/) was utilized for GO annotation. First, obtained protein identifications were mapped to GO IDs based on their uniport IDs. For proteins with no annotation in UniProt-GOA, InterProScan was utilized for annotating GO functions based on the protein sequence alignment. Proteins were assigned to biological process, cellular component and molecular function as GO terms. In various categories, a twotailed Fisher's test was utilized for assessing the enrichment of differentially expressed proteins versus all detected proteins; corrected P<0.05 indicated statistical significance.

Subcellular localization
Wolfpsort, latest version of PSORT/PSORT II, was utilized for predicting the subcellular localization of eukaryotic proteins.

BAF155-TG mice
For the biological repeatability of omics results, we determined that the quantitative data
Frozen myocardial tissue sections were examined by immunofluorescence. Crosssectional areas of cardiomyocytes were assessed in images obtained after staining with 5 µM wheat germ agglutinin (WGA) (Thermo, USA).

Echocardiography and left ventricular function (LVEF) assessment
Heart function in the Myh6

Micro-CT
Cardiac cavity was assessed by the section of the mean heart tissue brightness following contrast agent injection into vein. Micro-computed tomography (micro-CT-Imaging skycan 1276, Bruker, Germany) was performed at 55 kV (200 μA), acquiring 280 sections (13.275mm×13.275mm) in 2 min 51 s with steply ro-tating (degree=0.8). The CT images of the heart were opened with data viewer software, and the left ventricle was divided equally at both ends near the apical and the atrial part, and eight cross-sections with equal interval distance between them were taken as the results of our continuous cardiac scans to assess the area of the heart chambers.

Blood pressure measurement
Blood pressure (BP) was measured in conscious mice by the tail-cuff BP measurement system (BP-2010 Series Blood Pressure Meter, Softron, Japan). The blood pressure machine was prepared and preheated to 37℃ in advance, then the sensor was put on the tail of the mouse, and the mouse was placed in a mouse holder for 5 min to acclimate. Next, the blood pressure in the tail was measured three times per mouse. The average of the three pressure measurements represents the recorded mouse blood pressure.

Statistical analysis
Data are shown as mean±standard deviation (SD). Homogeneity of variance was assessed by the F-and Brown-Forsythe tests for two and multiple groups, respectively. The Shapiro-Wilk test was performed for assessing normality. Data with normal and skewed distributions were compared by Student's t test and Welch's t-test, respectively for two groups. One-way ANOVA and two-way ANOVA were carried out for multiple group comparisons involving one and two parameters, respectively, followed by post hoc Bonferroni test. The P values were appropriately adjusted for multiple comparisons.  Figure S1. BAF155 is up-regulated in failing hearts and cardiomyocytes, myocardium-specific BAF155 knockout significantly relieves cardiac hypertrophy and fibrosis in mice a Representative immunoblotting analysis of BAF155 in WT hearts after 2 weeks of Ang II infusion (1.5 mg/kg/day) and quantification of the relative BAF155 protein level. Data are mean±SD (***P<0.001; n=6/group) b Representative immunoblotting analysis of BAF155 in H9c2 cells exposed to Ang II (100 nM) for 24h and quantification of the relative BAF155 protein level (***P<0.001; n=6/group). c Representative expression levels of BAF155 were examined by immunofluorescence staining. The mouse heart tissues were labeled with anti-BAF155 antibodies (red), and nuclei were labeled with DAPI (blue). Scale bar, 50 µm. Data are mean±SD (***P<0.001; n=6/group). d Representative immunoblotting analysis of BAF155 in the BAF155-cWT/cKO mouse heart tissues after 2 weeks of Ang II infusion (1.5 mg/kg/day) and quantification of the relative BAF155 protein level. Data are mean±SD (*P<0.05, n=6/group). e, f, g Heart weight/body weight (HW/BW) (e) and heart weight/tibia length (HW/TL) (f) ratios, and representative immunoblotting (g) analysis of ANP, BNP and Cleaved caspase-3, were used to quantitate myocardial hypertrophy. Data are mean±SD (**P<0.01, ***P<0.001; n=6/group). h Representative immunoblotting analysis of α-SMA and Col-1 was used to quantify myocardial fibrosis. Data are mean±SD (*P<0.05, ***P<0.001; n=6/group). i Representative immunoblotting analysis of Cleaved-caspase-9 was used to quantify and cardiomyocyte death. Data are mean±SD (**P<0.01; n=6/group). j Representative immunoblotting analysis of p-ATM and p-ATR was used to quantify DNA damage. Data are mean±SD (**P<0.01, ***P<0.001; n=6/group). Data are expressed as means ± SD. Statistical significance was assessed by 2-way ANOVA with Bonferroni multiple comparisons test (P values adjusted for 6 comparisons, * P < 0.05; ** P < 0.01; *** P < 0.001). Figure S2. myocardium-specific BAF155 knockout significantly relieves cardiac hypertrophy and vascular thickness and fibrosis in mice a Plane and selected 3D reconstruction images were used to analyze the cardiac hypertrophy in BAF155-cWT mice and BAF155-cKO mice after 2 weeks of Ang II infusion (1.5 mg/kg/day). and ubiquitin sites (c) overlapping in mouse heart tissues between the WWP2-cKO and WWP2-cWT groups. d Column chart showing the distribution of ubiquitin sites for each protein with differential expression. e Column chart showing the distribution of ubiquitin sites identified in heart tissues from WWP2-cKO and WWP2-cWT mice, respectively. f The differentially ubiquitinated proteins in the hearts of WWP2-cKO and WWP2-cWT mice were enriched by biological process and represented by a bubble graph. The bubble color and size represent Fisher's exact test P value and the amounts of proteins with corresponding functions, respectively (n=3 per group). g The differentially ubiquitinated proteins in the hearts of WWP2-cKO and WWP2-cWT mice were enriched by molecular function and represented by a bubble graph. The bubble color and size represent Fisher's exact test P value and the amounts of proteins with corresponding functions, respectively (n=3 per group). h The differentially ubiquitinated proteins in the hearts of WWP2-cKO and WWP2-cWT mice were predicted and classified for subcellular structural mapping, (n=3 per group). i WWP2-cWT and WWP2-cKO heart tissues were obtained from mice with or without Ang II treatment, and underwent immunoprecipitation with anti-BAF155 antibodies, followed by detection with anti-PARP1 antibodies. j WWP2-cWT and WWP2-cKO heart tissues were obtained from mice with or without Ang II treatment, and ubiquitinated PARP1 underwent immunoprecipitation with anti-PARP1 antibodies, followed by detection with anti-UB antibodies. k WWP2-cWT and WWP2-cKO heart tissues were obtained from mice with or without Ang II treatment, and ubiquitinated BAF155 underwent immunoprecipitation with anti-BAF155 antibodies, followed by detection with anti-UB antibodies.